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J Tradit Chin Med 2013 April 15; 33(2): 233-237 ISSN 0255-2922 2013 JTCM. All rights reserved.

EXPERIMENTAL STUDY TOPIC

Effect of Zishenshengxue capsule on myelosuppression in mice induced by cyclophosphamide

Wei Li, Ying Zhao, Xinna Li aa

Wei Li, Ying Zhao, Xinna Li, Department of Pharmacy, Harbin Commercial University, Harbin 150076, China Supported by Heilongjiang Science and Technology Program (No. 2004 G0076-00) Correspondence to: Prof. Ying Zhao, Department of Pharmacy, Harbin Commercial University, Harbin 150076, China. wangwei99408131@163.com Telephone: +86-451-84883451 Accepted: October 16, 2012

0.01) compared with the myelosuppression group. C-GMs of middle and high dose ZSC groups significantly increased (P<0.01). The percentage of G1 phase in the high dose ZSC group decreased (P< 0.01) and the percentage of S and G2 phase increased (P<0.01). CONCLUSION: ZSC increased the numbers of peripheral white blood cells, bone marrow karyocytes and C-GMs. ZSC also increased cell proliferation activity and removed the G1 phase block. Thus, ZSC could reduce myelosuppression in mice induced by cyclophosphamide.

Abstract
OBJECTIVE: To explore the inhibitory effect of Zishenshengxue capsule (ZSC) on myelosuppression in mice induced by cyclophosphamide. METHODS: Kunming mice were randomly assigned into a control group, myelosuppression group, or groups for mice with myelosuppression receiving high dose ZSC, middle dose ZSC, low dose ZSC or Yixuesheng. Myelosuppression was induced by peritoneal injection of cyclophosphamide. ZSC and cyclophosphamide were administered simultaneously. The numbers of peripheral blood cells and bone marrow karyocytes were counted. Cell proliferation activity and colony formation of granulocyte-monocyte series hemopoietic progenitor cells (C-GMs) and cell cycle were detected. RESULTS: The numbers of white blood cells in the middle and high dose ZSC groups were significantly increased at the 12th and 13th day (P<0.01) and bone marrow karyocytes and cell proliferation activity increased in the high dose ZSC group (P<
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2013 JTCM. All rights reserved. Key words: Myelosuppression; Cell proliferation; Cell cycle; Zishenshengxue capsule

INTRODUCTION
For cancer patients, chemotherapy is the optimal therapy available. However, chemotherapy causes myelosuppression that hampers the beneficial effects of the treatment and decreases the clinical effect.1 Therefore, the development of methods that relieve myelosuppression and promote the recovery of hematogenesis to increase the peripheral white blood cells is a key problem to the successful completion of chemotherapy and clinical effects. Zishenshengxue capsule (ZSC) is a Chinese herbal compound that can treat bone marrow depression induced by numerous factors especially chemotherapy and has valid clinical effect.2 In this study, we investigated the inhibitory effects of ZSC on myelosuppression in mice induced by cyclophosphamide and suggest a mechanism of action.
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MATERIALS AND METHODS

Drugs and reagents ZSC was provided by the Harbin Institute of Hematology and Tumor. Granulocyte and monocyte colony stimulating factor (GM-CSF) was purchased from Stem Cell Technologies (Vancouver, Canada). Cyclophosphamide (CTX) was purchased from Hua Lian Company (ShangHai, China). Yixuesheng (YXS, positive control), (positive control) was purchased from AoDong Company (JiLin, China). RPMI-1640 cell culture medium was purchased from Gibco (Carlsbad, CA, USA). Animals Kunming mice [male, (202) g] were provided by the Experimental Animal Center of Harbin Medical University (certificate serial number 09-2-1). This research was approved by the animal ethics committees of Department of Pharmacy, Harbin Commercial University. Preparation of mice skeletal muscle conditioned medium Normal Kunming mice were euthanized by cervical vertebra dislocationandthenplacedin75%ethanol for 5 min twice. The back muscles and both lower extremities muscles were dissected and cut into 0.3 cm3 pieces, rinsed with RPMI-1640 media and then incubated for 2 h. One g muscle was placed in 9 mL media containing 2 mL horse serum (HS) and 7 mL RPMI-1640. The muscles were then cultured at 37 C in 5% CO2 for 7 days. The supernatant was collected and centrifuged for 5 min at 4C at 500 g and stored at 20C after filtration until used.3 Detection of peripheral blood cells Mice (n=84) were randomly assigned into 6 groups according to weight, 14 per group as follows: control group (control), the model myelosuppression group (model), high dose ZSC (6.6 g/kg per day) group (H), middle dose ZSC (3.3 g/kg per day) group (M), low dose ZSC (1.65 g/kg per day) group (L) and YXS (0.375 g/kg per day) group (a positive control). The mice in the control group were given saline by peritoneal injection and myelosuppression was induced in the other groups by peritoneal injection of CTX (50 mg/ kg) on the 1st and 3rd day. The H, M, L and YXS group were simultaneously administered either ZSC or YXS by intragastric administration for 12 days. The mice in the control and model group were administered saline by intragastric administration (0.2 mL per day). Peripheral blood obtained from the tails of each group was used to measure leukocyte, erythrocyte and platelet numbers on days 4, 5, 6, 8, 12 and 13. Counting of bone marrow karyocytes Another 60 mice were manipulated as above. On day
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13, mice were euthanized by cervical vertebra dislocation and then placed in 75% ethanol for 5 min twice. Double femurs were removed under aseptic conditions and bone marrow cells were flushed using 1640 medium to form a single cell suspension. The suspension was placed into a tube containing lymphocyte separation medium, and centrifuged for 5 min at 2000 g. The bone marrow karyocytes were removed and rinsed twice with 1 mL sterile phosphate-buffered saline (PBS) for 5 min at 1000 g then resuspended in RPMI 1640 and counted by inverted microscope.4 Cell viability analysis The bone marrow karyocytes (2.5105/mL) were resuspended in RPMI-1640 medium containing 20% HS and 10% mouse skeletal muscle conditioned medium. These cells were placed into 96-well plates in triplicate and incubated at 37C, in a 5% CO2 incubator for 48 h. 3,[4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide (MTT, 5 mg/mL)5 was then added to each well. Then cells were incubated for another 4 h. The supernatants were discarded and 180 L DMSO was added to each well and oscillated for 10 min. The optical density (OD) was measured at 570 nm. Colony formation of granulocyte-monocyte series hemopoietic progenitor cells (C-GMs) Cells (5104) were plated in triplicate cultures in 1 mL of methylcellulose assay medium containing HS (0.2 mL) and GM-CSF (100 ng/mL). The cells were cultured at 37 C, in a 5% CO2 incubator for 7 days. Colonies of 40 or more cells were counted as colonies, using a inverted-phase microscope. 6 Cell cycle A single cell suspension was collected into a centrifuge tube and rinsed twice with cold PBS, centrifuged and then 2 mL 70% cold ethanol was added to fix the cells overnight at 4C. Cells were then incubated with propidium iodide (PI) in the dark for 30 min. Cells from all groups were measured by flow cytometry (FANCSCanto , BD company, New Jersey, USA). 7 Statistical analysis The data were expressed as mean standard deviation. Statistical analysis was performed by one-way analysis of variance. All analyses were performed with SPSS 13.0 statistical software (SPSS Inc. Chicago, IL, USA) and P<0.05 was considered statistically significant.

RESULTS
Effect of ZSC on peripheral blood cells As shown in Figure 1, the number of leukocytes in the model group significantly decreased compared with the control group (P<0.01) at days 4, 5 and 6. However, compensation mechanisms caused an increase in the
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c d

leukocyte (109)

c a a a a a b c

Figure 1 Effect of ZSC on peripheral leukocyte Control: control group; model: the model myelosuppression group; H: high dose ZSC group; M: middle dose ZSC group; L: low dose ZSC group; YXS: YXS group. ZSC: Zishenshengxue capsule; YXS: Yixuesheng. Data are expressed as the mean SD (n=14 rats/ group). aP<0.01, bP<0.05, vs control; cP<0.05, dP<0.05, vs model.

number of leukocytes in the model group by day 10. On days 12 and 13 the leukocyte numbers in the H, M, L and YXS groups were significantly increased (P< 0.01) compared with the model group. The number of erythrocytes in the model group did not change compared to the control group and administration of ZSC or YXS had no effect on erythrocyte numbers or platelets (Supplementary file). Effect of ZSC on bone marrow karyocytes Bone marrow karyocytes mainly include immature cells in bone marrow such as total leukocytes, megakaryocytes and immature erythrocytes. As shown in Figure 2, numbers of karyocytes in the model group significantly decreased compared with the control group (P<0.01). The high dose of ZSC significantly increased karyocyte numbers compared with the model group (P<0.05) and its effect was superior to YXS.
b a c

(P<0.01). The bone marrow cell proliferation significantly increased after the administration of high dose of ZSC and YXS compared with the model group (P< 0.05). As shown in Figure 4, the C-GMs numbers in the model group significantly decreased compared with the control group (P<0.01). Compared with the model group, C-GMs numbers in L, M, H and YXS groups significantly increased (P<0.05). Effect of ZSC on cell cycle The ratio of G1 phase in the model group increased and the ratio of S and G2 phase decreased after CTX administration (Figure 5). These changes in cell cycle phase were significant compared with the control group (P<0.05). The percentage of G1 phase cells in H and YXS groups significantly decreased (P<0.01) and S phase cells significantly increased (P<0.01) compared with the model group.

DISCUSSION
Chemotherapy restricts the effect of tumor treatment by causing myelosuppression and is an important problem that must be solved to optimize chemotherapy for the treatment of cancer patients. Clinical studies demonstrated that Batiolum and Leucogen have little effect on myelosuppression. Although many cytokines can stimulate cell differentiation and proliferation as well as promote the recovery of hemogram and elevate clinical effects, some side effects may occur because of the large dosage required. Autologous bone marrow transplantation does not satisfy clinical need due to its expensive cost and non-repetitive application. Therefore, it is challenging to identify drugs from Traditional Chinese Medicine or Western Medicine that have high therapeutic efficacy with low toxicity and side effects and that are relatively cheap. In this study, we used CTX peritoneal injection to in235 April 15, 2013 | volume 33 | Issue 2 |

Control Model L M H YXS Figure 2 Effect of ZSC on bone marrow karyocyte Control: control group; model: the model myelosuppression group; H: high dose ZSC group; M: middle dose ZSC group; L: low dose ZSC group; YXS: YXS group. ZSC: Zishenshengxue capsule; YXS: Yixuesheng. Data are expressed as the meanSD (n=10 rats/group). aP<0.01, bP<0.05, vs control; cP< 0.05 vs model.

Effects of ZSC on bone marrow cell proliferation As shown in Figure 3, there was significant reduction in bone marrow cell proliferation as measured by OD in the model group compared with the control group
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c a b

Control Model

YXS

Figure 3 Effect of Zishen Shengxue capsule on bone marrow cell proliferation activity Control: control group; model: the model myelosuppression group; H: high dose ZSC group; M: middle dose ZSC group; L: low dose ZSC group; YXS: YXS group. ZSC: Zishenshengxue capsule; YXS: Yixuesheng; OD: optical density. Data are expressed as the mean SD (n=10 rats/group). aP<0.01, bP< 0.05, vs control; cP<0.05 vs model.

c a

Control Model L M H YXS Figure 4 Effects of ZSC on C-GMs Control: control group; model: the model myelosuppression group; H: high dose ZSC group; M: middle dose ZSC group; L: low dose ZSC group; YXS: YXS group. ZSC: Zishenshengxue capsule; YXS: Yixuesheng; C-GMs: colony formation of granulocyte-monocyte series hemopoietic progenitor cells. Data are expressed as the meanSD (n=10 rats/group). acP< 0.01 vs control; cP<0.05 vs model.
a

a a

b b

Control Model

YXS

duce a mouse model of bone marrow depression and studied the effect of ZSC administration on myelosupJTCM | www. journaltcm. com

Figure 5 Effects of ZSC on cell cycle Control: control group; model: the model myelosuppression group; H: high dose ZSC group; M: middle dose ZSC group; L: low dose ZSC group; YXS: YXS group. ZSC: Zishenshengxue capsule; YXS: Yixuesheng. Data are expressed as the mean SD (n=10 rats/group). aP<0.01 vs control; bP<0.05 vs model.

pression. After CTX injection, these mice showed a weak mental state and retarded action with fur that was dull, withered and disordered. In addition, the number of leukocytes decreased after CTX injection. However, the number of leukocytes increased compensatively by day 10. This may be due to a positive feedback loop, where the killing of blood cells in the multiplicative pool by CTX induced the proliferation of hemopoietic stem cells in the non-multiplicative pool. Thus, large numbers of leukocytes may mature synchronically and enter the blood stream, thus reducing the number of bone marrow leukocytes. The number of bone marrow karyocytes and proliferation activity also significantly decreased. These characteristics are similar to the symptoms of cancer patients treated with chemotherapy. Therefore, this model may simulate the myelosuppression state of tumor patients after chemotherapy and may be useful for the study of the preventive and therapeutic effects of ZSC. Hematocytopenia describes major bone marrow depression and hypoleukemia is more notable because of its short half-life of approximately 6-8 h. However, the half-life of platelets and erythrocytes are relatively longer, so obvious changes are not usually observed. Leukocytes increased significantly in mice on days 12 and 13 after administration of low, middle and high doses of ZSC, and the effect of high dose ZSC was most significant. Concurrently, bone marrow karyocytes nearly recovered to normal levels, which reflects the proliferation state of the bone marrow cells. The bone marrow cell viability and C-GMs increased significantly in the ZSC groups' indicating that the number of living cells and internal proliferation activity increased. Cells in G1 phase increased notably after CTX induction and proliferation and differentiation were inhibited. Therefore, the later G1 phase is a key point for the promotion of the cell cycle and also a target for drugs and other factors that affect the cell cycle.8 How to increase the number of hematopoietic cells that pass the G1 phase checkpoint and enter S phase to recover impaired hematogenesis function of bone marrow by chemotherapy is an important problem in drug studies. 9 High dose ZSC decreased the ratio of G1 phase cells and elevated the percentage of cells in S and G2 phase. Thus, ZSC could promote cells to pass the G1/S phase checkpoint and successfully enter the cell cycle to facilitate the repair of damaged DNA and accelerate cell proliferation. Therefore, ZSC treatment antagonized the toxicity and side effects of CTX on bone marrow cells. In summary, ZSC could reduce bone marrow lesions caused by chemotherapy and reduce CTX-induced myelosuppression in mice. Furthermore, ZSC was superior to YXS, a commonly used drug, at increasing the number of bone marrow karyocytes.

C-GMs

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